The present invention relates to a device for separating substances of high molecular weight from a mixture. This invention in particular relates to a device for carrying out dialysis, purification, desalting or concentration of proteins. This invention, preferably, proposes a device which is simple to operate, manufacture and offers easy loading and recovery of protein samples.
Dialysis is one of the most commonly utilized methods for transferring a biological sample, usually protein based, from one media to another. It is frequently necessary to remove salts or change the buffer after one step in the purification for the next step to work efficiently (e.g. in ion-exchange chromatography, the pH and/or the ionic strength may have to be changed to ensure that the protein will bind to the matrix). This is often achieved by dialysis: wherein the protein solution kept in a semi-permeable membrane is placed in the required buffer so that small molecules e.g. salts, can pass freely across the membrane whilst large molecules e.g. proteins, are retained. From the removal of salts to the exchange of buffer systems, dialysis tubing has established itself as the method of choice because it is quick and simple to use, readily available and relatively inexpensive. The semi-permeable dialysis tubing is usually made of cellulose acetate having pores of approximate size of 1-20 nm in diameter. The size of these pores determines the minimum molecular weight of molecules which will be retained by the membrane (NMWC=molecular weight cut off). Dialysis tubing often requires pre-treatment to ensure a more uniform pore size and removal of heavy metal contamination.
Traditional dialysis or desalting i.e. protein purification methods involving use of devices such as semipermeable membrane tubing are cumbersome, involving boiling, soaking, tying or clamping of membrane, which are time and effort consuming. They also result in loss of precious protein sample which loss becomes significant when sample volume is small during processing. There are various devices available for dialysis in the market manufactured by M/s.Millipore, Amicon, Pierce and Sartorium. Improved, modem methods employ tubes, having membranes fitted inside them. When these tubes, loaded with sample and are centrifuged, the smaller particles e.g. salts, low molecular weight contaminants etc. pass through, while the larger particles i.e. the proteins are retained. Thus, purification or desalting of proteins is achieved. By employing membranes of different cut-off values, proteins of different molecular weights can also be separated. The process though quick and efficient, has many limitations. For example, these tubes cannot be operated without a centrifuge, and as dialysis/purification of proteins is usually carried out at 4xc2x0 C., a refrigerated centrifuge becomes essential. The utility of such methods thus becomes limited owing to essential requirement of an external, expensive piece of equipment for operation. Further, in event of interruption in the power supply, these devices cannot be utilized at all. Furthermore, the semi-permeable membranes fitted inside tubes are of a fixed volume, which restricts flexibility regarding the volume of the sample which can be processed in a single unit. Such units are quite expensive, disposable type and not capable of being reused.
U.S. Pat. No. 5,324,428 provides a disposable dialysis apparatus suspended in a dialysate solution. The dialysis apparatus has a tubular membrane which is opened at the top and bottom. A closed base membrane and collar seals the bottom of the tubular membrane. An open upper membrane and collar seals the top of the tubular membrane, and a cap may be affixed to the top of the open upper member to seal the contents. This device is rather cumbersome to operate especially when sample volumes are small e.g. less than 15 ml. Also, it is costly to manufacture and requires semi-permeable membrane in tubular form which needs pretreatment e.g. boiling, before use.
U.S. Pat. No. 5,342,517 provides a filtration cassette comprising membrane filter sheets arranged in a peripherally bonded array of multilayered character wherein the filter sheets alternate with foraminous (e.g., screen or mesh) sheet elements and a cross-flow filter comprising a multiplicity of stacked filtration cassettes of such type. The device is a complicated one, suited as an on-line device for dialysis of a flowing solution or a single solution. The multiplicity of such units required when a large number of protein samples have to be processed make it unsuitable for routine laboratory use.
U.S. Pat. No. 5,503,741 provides a device for dialysis with hermetically sealed vacant chamber. The said device consists of a semi-permeable membrane sealed in a plastic frame. Sample is loaded and recovered with help of a syringe. The device though an improvement over traditional methods of dialysis, has several limitations. Firstly, the semi-permeable membrane is fixed in a frame, which restricts flexibility regarding volume of sample to be processed i.e. cassettes are of fixed size. For larger volumes, multiple cassettes have to be used for same sample or larger sized cassettes are needed. Secondly, sample loading as well as recovery, involves use of syringe and needle which causes sample loss due to sticking The sample losses can be significant when handling small volumes since the sample sticks to the walls of the gasket and syringe. In case of multiple samples, a number of syringes equal to number of samples, are required which makes the loading and recovery of sample, time consuming, tedious and expensive besides posing the risk of accidentally puncturing the membrane or causing injury to user. Thirdly, the cassettes are not reusable, and the entire unit has to be discarded after single use. Fourthly, removal of membrane protecting agent viz. glycerol, from the membrane is quite cumbersome requiring injection of distilled water into the cassette and running it in distilled water for a minimum of four hours. Lastly, the cassettes are quite expensive and cannot be reused, which is a limitation requiring special manufacturing facilities owing to nature of their design.
As such in order to overcome the drawbacks of the hitherto known devices for dialysis, the applicants have invented the present device for dialysis, desalting, concentration or purification of proteins. The said device comprises of three parts viz. upper hollow chamber, middle detachable air-tight seal and lower semipermeable sac, the upper and lower parts being connected to each other by one or more apertures located in the detachable air-tight seal.
The main object of the present invention is to provide a simple and cost-effective device for dialysis, desalting, concentration or purification of proteins.
Another object of the invention is to provide a device for dialysis of proteins which is easy to manufacture and easy to operate.
Yet another objective of the invention is to provide a device for dialysis which offers considerable flexibility regarding volume of the sample to be processed.
Still another object of the invention is to provide a device that can be reused for the same sample or for different samples.
Another object of the present invention is to provide a device for dialysis wherein sample loading and unloading is extremely easy without any loss of sample.
One more object of the present invention is to provide a device for dialysis wherein no external equipment such as centrifuge or syringe is used and the process sample remains untouched by hand.
To meet the above and other objects, the present invention provides a device for dialysis/desalting/purification/concentration of proteins, consisting of three parts viz. upper hollow float chamber, middle detachable, air-tight seal and lower semipermeable sac, the upper and lower parts being connected to each other by an aperture in the detachable air-tight seal.
The hollow float chamber can be made of any suitable shape but preferably cylindrical and made of any suitable material such as rigid or flexible plastic, glass or rubber. The hollow chamber can be made of transparent material sample to see the sample and the chamber can be graded/graduated for easy volume measurement.
The detachable air-tight seal attached to the hollow chamber, is preferably circular, made of any suitable material such as rigid or flexible plastic and rubber and has one or more apertures which may or may not be centrally located The sac of semipermeable membrane is detachably attached to the air-tight seal.
The protein sample to be purified is put into the semi-permeable sac via the aperture in the seal, and seal is then attached to hollow chamber in any conventional manner, after which the device is floated in a tank of buffer/distilled water, for subjecting the sample to dialysis/purification/ desalting.
In a preferred embodiment, the device is placed in a concentrating solution e.g. Polyethylene glycol (PEG), instead of water or buffer such as ammonium bicarbonate and water is drawn out through the semi-permeable membrane, resulting in the protein sample getting concentrated.
The purified/concentrated sample is then recovered by removing the device from the tank and inverting to device so that the sample drains from sac into the hollow float chamber, which is then opened by detaching air-tight seal, for recovering sample.
The invention is described with reference to the accompanying drawings and it should not be construed to limit the scope of the invention.